Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

A molecular basis for nitric oxide sensing by soluble guanylate cyclase

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (kon > 1.4 × 108 M−1⋅s−1 at 4°C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k = 2.4 × 105 M−1⋅s−1 at 4°C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k = 7.1 ± 2 × 108 M−1⋅s−1 at 4°C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Guanylyl cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues

Guanylyl cyclase C (GCC) has been detected only in intestinal mucosa and colon carcinoma cells of placental mammals. However, this receptor has been identified in several tissues in marsupials, and its expression has been suggested in tissues other than intestine in placental mammals. Selective expression of GCC by colorectal tumor cells in extraintestinal tissues would permit this receptor to be employed as a selective marker for metastatic disease. Thus, expression of GCC was examined in human tissues and tumors, correlating receptor function with detection by PCR. GCC was detected by ligand binding and catalytic activation in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues or tumors. Similarly, PCR yielded GCC-specific amplification products with specimens from normal intestine and primary and metastatic colorectal tumors, but not from extraintestinal tissues or tumors. Northern blot analysis employing GCC-specific probes revealed an ≈4-kb transcript, corresponding to recombinant GCC, in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues. Thus, GCC is selectively expressed in intestine and colorectal tumors in humans and appears to be a relatively specific marker for metastatic cancer cells in normal tissues. Indeed, PCR of GCC detected tumor cells in blood from some patients with Dukes B colorectal cancer and all patients examined with Dukes C and D colorectal cancer, but not in that from normal subjects or patients with Dukes A colon carcinoma or other nonmalignant intestinal pathologies.

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in Aplysia sensory neurons

Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa1, that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

The α1- and β1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Genomic organization of α1 and β1 subunits of the mammalian soluble guanylyl cyclase genes

The structures of the genes encoding the α1 and β1 subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the α1 (2.5 kb) and β1 (3.3 kb) subunits are presented in this report. The α1 sGC gene is approximately 26.4 kb and contains nine exons, whereas the β1 sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse α1 and β1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5′ untranscribed regions of α1 and β1 subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5′ untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Gαs stimulation

Receptors activate adenylyl cyclases through the Gαs subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Gαs. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Gαs. Similar treatment of AC1 or AC2 did not affect Gαs stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Gαs stimulation. A peptide encoding the region 660–682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Gαs. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660–682 of AC6 is involved in regulation of signal transfer from Gαs. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.

A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.